Human immunodeficiency virus type 1 (HIV-1) infection is initiated when the surface envelope (ENV) glycoprotein of the virion binds to specific receptors on the surface of an appropriate target cell. These cell surface receptors are CD4 and a chemokine receptor coreceptor. The discovery of the coreceptor molecules has provided new insights into the mechanism of virus entry and has offered new directions for the development of antiviral therapies and vaccines. The production of a broadly effective vaccine based on Env is greatly complicated by the existence of multiple HIV-1 genotypes. HIV-1 Env-based subunit vaccines have thus far failed to efficiently generate neutralizing antibodies that are broadly cross-reactive. One possible explanation for this failure is that the present formulations of soluble Env do not present the best neutralizing epitopes. Previously, we hypothesized that the first step in the virus entry process would be the formtion of a trimolecular complex between the HIV-1 Env, CD4 and a coreceptor and that membrane fusion could be correlated to the formation of such a complex, and recently we have provided evidence supporting this model. A long-standing notion of this interaction between Env and its receptors, and the ensuing conformational changes preceding fusion, was that new epitopes would be exposed in the trimolecular complex and antibodies specific for this 'fusion- competent' intermediate could potentially neutralize virus by blocking entry. Most recently this notion was tested, and mixtures of chemically fixed HIV-1 Env-expressing and receptor expressing cells undergoing fusion were used as immunogens in CD4-CCR5 transgenic mice. The antibody responses generated from these mixtures possessed a broadly cross-reactive neutralizing activity and this proof-of-principle experiment has laid the ground work for developing a refined trimolecular/fusion- competent vaccine. Ths overall goal of this project is to further develop and evaluate the potential of novel Env-CD4- coreceptor complexes as immunogens for the elicitation of HIV-1 neutralizing antibody responses. Specifically, we will: 1) Prepare and characterize Env-CD4-coreceptor complexes generated in our laboratory, refine their method of preparation using affinity-based precipitation approaches (immunobead and Histine- Tag methodologies) and purify, quantify and determine the stability of the prepared complexes. 2) Immunize CD4-CCR5 transgenic mice with Env-CD4-coreceptor complexes and characterize the elicited antibodies by assessing their neutralization activity and cross-reactivity to a variety of HIV- 1 isolates. 3) Generate mouse monoclonal antibodies to Env-CD4- coreceptor complexes and determine if complex-specific epitopes can be identified and mapped and whether they possess HIV-1 neutralizing activity.